Effect of age and cytoskeletal elements on the indentation-dependent mechanical properties of chondrocytes.

Nadeen O Chahine,Craig Blanchette,Cynthia B Thomas,Dominik Haudenschild,Jeffrey Lu,Gabriela G Loots,Laurent Kreplak

doi:10.1371/journal.pone.0061651

Abstract

Articular cartilage chondrocytes are responsible for the synthesis, maintenance, and turnover of the extracellular matrix, metabolic processes that contribute to the mechanical properties of these cells. Here, we systematically evaluated the effect of age and cytoskeletal disruptors on the mechanical properties of chondrocytes as a function of deformation. We quantified the indentation-dependent mechanical properties of chondrocytes isolated from neonatal (1-day), adult (5-year) and geriatric (12-year) bovine knees using atomic force microscopy (AFM). We also measured the contribution of the actin and intermediate filaments to the indentation-dependent mechanical properties of chondrocytes. By integrating AFM with confocal fluorescent microscopy, we monitored cytoskeletal and biomechanical deformation in transgenic cells (GFP-vimentin and mCherry-actin) under compression. We found that the elastic modulus of chondrocytes in all age groups decreased with increased indentation (15–2000 nm). The elastic modulus of adult chondrocytes was significantly greater than neonatal cells at indentations greater than 500 nm. Viscoelastic moduli (instantaneous and equilibrium) were comparable in all age groups examined; however, the intrinsic viscosity was lower in geriatric chondrocytes than neonatal. Disrupting the actin or the intermediate filament structures altered the mechanical properties of chondrocytes by decreasing the elastic modulus and viscoelastic properties, resulting in a dramatic loss of indentation-dependent response with treatment. Actin and vimentin cytoskeletal structures were monitored using confocal fluorescent microscopy in transgenic cells treated with disruptors, and both treatments had a profound disruptive effect on the actin filaments. Here we show that disrupting the structure of intermediate filaments indirectly altered the configuration of the actin cytoskeleton. These findings underscore the importance of the cytoskeletal elements in the overall mechanical response of chondrocytes, indicating that intermediate filament integrity is key to the non-linear elastic properties of chondrocytes. This study improves our understanding of the mechanical properties of articular cartilage at the single cell level.

Highlights

Articular cartilage is the connective tissue that lines the ends of bones in diarthrodial joints and provides a low-friction bearing surface for the transmission and distribution of mechanical loads in the skeleton
Our findings indicate that the elastic modulus of chondrocytes isolated from different aged bovines decreases with increasing deformation
We systematically measured the contributions of actin and intermediate filaments (IFs) on the indentation dependent mechanical properties of chondrocytes isolated from 1 day old bovine joints using cytochalasin B (cytoB) and acrylamide, respectively

Summary

Introduction

Articular cartilage is the connective tissue that lines the ends of bones in diarthrodial joints and provides a low-friction bearing surface for the transmission and distribution of mechanical loads in the skeleton. Chondrocytes are the dominant cell type found in articular cartilage, and these cells are responsible for the synthesis, maintenance, and gradual turnover of the extracellular matrix (ECM) [1]. The cartilage ECM has been shown to remodel in response to the functional demands of mechanical loading, and this process is mediated through the metabolic activity of chondrocytes [1]. Articular cartilage is a functionally heterogeneous tissue such that the position of a chondrocyte relative to the joint surface and the bone interface defines its extracellular and intracellular biochemical composition [4,5] resulting in zonal depth-dependent mechanical properties [6,7,8,9,10]. Certain zonal changes in metabolic activity have been correlated with zonal strain magnitudes, suggesting that zone-specific variations in mechanical stimuli could be responsible for spatially varied patterns of cartilage metabolic activity under load [19]

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